2629 Short Report Introduction ESCs derived from the inner cell mass of the blastocyst can differentiate into primitive ectoderm, primitive endoderm and trophoectoderm cells, and in turn into all cell types present in the embryo. ESCs are maintained in the undifferentiated state during self-renewal by a complex regulatory network involving three transcription factors, namely Oct3/4 (Pou5f1) (Nichols et al., 1998), Nanog (Chambers et al., 2003; Mitsui et al., 2003) and Sox2 (Avilion et al., 2003). These factors regulate their own expression and that of many other genes (Boyer et al., 2005; Loh et al., 2006). Other transcription factors play important roles in ESC pluripotency and self-renewal (Niwa, 2007). These data illustrate the complexity of transcription regulation in ESCs, which is still not completely understood. Recently, several results indicate that transcription factors belonging to the Kruppel-like family could have an important role in the regulation of ESCs. Ectopic expression of Klf4, together with Oct3/4, Sox2 and Myc, results in the conversion of differentiated cells into pluripotent ES-like cells (Takahashi et al., 2006; Okita et al., 2007; Werning et al., 2007; Maherali et al., 2007). Recent findings indicate that Klf2 or Klf5 can replace Klf4 in the gene combination inducing cell reprogramming (Nakagawa et al., 2008) and that triple knockdown (KD) of Klf2, Klf4 and Klf5 abolishes the undifferentiated phenotype of ESCs (Jiang et al., 2008). In this study, we demonstrate that KD of even only Klf5 abolishes the ESC undifferentiated phenotype, whereas its constitutive expression prevents ESC differentiation. Results and Discussion Klf5 is expressed in mouse ESCs and its knockdown suppresses normal ESC differentiation The screening of a collection of short hairpin RNAs (shRNAs) designed to target mouse mRNAs allowed us to observe that the shRNA targeting Klf5 mRNA was able to interfere with ESC differentiation (see supplementary material Fig. S1). Klf5, also known as intestinal-enriched factor and basic transcription element binding protein 2, is a Zn-finger transcription factor belonging to the Sp/Kruppel-like family. In adults, it is expressed in the proliferating crypt cells of the intestinal epithelium and at low levels in the testis, uterus, placenta, lung and in the proliferating basal layer of the epidermis (Ohnishi et al., 2000). Klf5 knockout causes early embryonic lethality (Shindo et al., 2002), which suggests that this factor plays a key role during early development. Klf5 mRNA and protein are present in undifferentiated ESCs and levels rapidly decrease after induction of ESC differentiation by two different approaches (Fig. 1A,B). Immunostaining demonstrated that Klf5 is expressed at various levels in almost all undifferentiated ESCs (Fig. 1C) and that it substantially colocalises with Oct3/4 and Nanog [964 out of 1100 cells (87.6%) were positive for both Oct3/4 and Klf5; 512 out of 678 cells (75.5%) were Nanog and Klf5 positive; 625 out of 720 cells (86.8%) expressed both Oct3/4 and Nanog]. After 3 days in differentiation conditions, the Klf5 signal disappeared from most cells, as observed for Oct3/4 and Nanog (supplementary material Fig. S2). The expression of Klf5 mRNA in vivo is in agreement with that observed during in vitro differentiation of ESCs. In fact, we found the Klf5 transcript Self-renewal of embryonic stem cells (ESCs) is maintained by a complex regulatory mechanism involving transcription factors Oct3/4 (Pou5f1), Nanog and Sox2. Here, we report that Klf5, a Zn-finger transcription factor of the Kruppel-like family, is involved in ESC self-renewal. Klf5 is expressed in mouse ESCs, blastocysts and primordial germ cells, and its knockdown by RNA interference alters the molecular phenotype of ESCs, thereby preventing their correct differentiation. The ability of Klf5 to maintain ESCs in the undifferentiated state is supported by the finding that differentiation of ESCs is prevented when Klf5 is constitutively expressed. Maintenance of the undifferentiated state by Klf5 is, at least in part, due to the control of Nanog and Oct3/4 transcription, because Klf5 directly binds to the promoters of these genes and regulates their transcription. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/16/2629/DC1 Key words: Differentiation, Kruppel-like factors, Nanog, Oct3/4, Self-renewal Summary Klf5 is involved in self-renewal of mouse embryonic stem cells Silvia Parisi 1,2, *, Fabiana Passaro 1,3, *, Luigi Aloia 1,2 , Ichiro Manabe 4 , Ryozo Nagai 5 , Lucio Pastore 1,3 and Tommaso Russo 1,3,‡ 1 CEINGE Biotecnologie Avanzate, 80145 Napoli, Italy 2 European School of Molecular Medicine, SEMM, 80145 Napoli, Italy 3 Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy 4 Nano-Bioengineering Education Program and 5 Department of Cardiovascular Medicine, The University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan *These authors contributed equally to this work ‡ Author for correspondence (e-mail: russot@dbbm.unina.it) Accepted 15 May 2008 Journal of Cell Science 121, 2629-2634 Published by The Company of Biologists 2008 doi:10.1242/jcs.027599 Journal of Cell Science